WO2014153052A2 - Cftr mrna compositions and related methods and uses - Google Patents

Cftr mrna compositions and related methods and uses Download PDF

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Publication number
WO2014153052A2
WO2014153052A2 PCT/US2014/028849 US2014028849W WO2014153052A2 WO 2014153052 A2 WO2014153052 A2 WO 2014153052A2 US 2014028849 W US2014028849 W US 2014028849W WO 2014153052 A2 WO2014153052 A2 WO 2014153052A2
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WIPO (PCT)
Prior art keywords
mrna
cftr
seq
coding sequence
lung
Prior art date
Application number
PCT/US2014/028849
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English (en)
French (fr)
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WO2014153052A9 (en
WO2014153052A3 (en
Inventor
Michael Heartlein
Braydon Charles Guild
Frank Derosa
Carsten Rudolph
Christian Plank
Lianne SMITH
Original Assignee
Shire Human Genetic Therapies, Inc.
Ethris Gmbh
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Priority to AU2014236305A priority Critical patent/AU2014236305B2/en
Priority to KR1020157023977A priority patent/KR102248744B1/ko
Priority to CA2904151A priority patent/CA2904151C/en
Priority to BR112015022868-2A priority patent/BR112015022868B1/pt
Priority to EP14718284.4A priority patent/EP2968586B1/en
Priority to SG11201507391VA priority patent/SG11201507391VA/en
Priority to MX2015012333A priority patent/MX365409B/es
Priority to EA201591477A priority patent/EA037922B1/ru
Priority to KR1020217012925A priority patent/KR102311614B1/ko
Priority to UAA201508754A priority patent/UA117008C2/uk
Priority to ES14718284.4T priority patent/ES2689523T3/es
Priority to CN202210265261.0A priority patent/CN115154620A/zh
Priority to PL14718284T priority patent/PL2968586T3/pl
Priority to CN201480015043.5A priority patent/CN105142676B/zh
Priority to JP2016502924A priority patent/JP6316930B2/ja
Priority to KR1020217031812A priority patent/KR20210122917A/ko
Priority to EP18185286.4A priority patent/EP3446712A1/en
Priority to LTEP14718284.4T priority patent/LT2968586T/lt
Priority to RS20181158A priority patent/RS57739B1/sr
Application filed by Shire Human Genetic Therapies, Inc., Ethris Gmbh filed Critical Shire Human Genetic Therapies, Inc.
Priority to NZ711657A priority patent/NZ711657B2/en
Priority to IL305374A priority patent/IL305374A/en
Priority to IL290953A priority patent/IL290953B2/en
Priority to SI201430888T priority patent/SI2968586T1/sl
Priority to DK14718284.4T priority patent/DK2968586T3/en
Priority to US14/307,322 priority patent/US9181321B2/en
Publication of WO2014153052A2 publication Critical patent/WO2014153052A2/en
Publication of WO2014153052A9 publication Critical patent/WO2014153052A9/en
Priority to IL240982A priority patent/IL240982B/en
Publication of WO2014153052A3 publication Critical patent/WO2014153052A3/en
Priority to US14/876,071 priority patent/US9713626B2/en
Priority to HK16105865.3A priority patent/HK1218068A1/zh
Priority to HK16108273.3A priority patent/HK1220137A1/zh
Priority to US15/625,648 priority patent/US10420791B2/en
Priority to HRP20181580TT priority patent/HRP20181580T1/hr
Priority to CY181101097T priority patent/CY1120790T1/el
Priority to IL262984A priority patent/IL262984B/en
Priority to AU2019202582A priority patent/AU2019202582B2/en
Priority to US16/540,960 priority patent/US11510937B2/en
Priority to IL282453A priority patent/IL282453B/en
Priority to AU2021215095A priority patent/AU2021215095B2/en
Priority to US18/049,210 priority patent/US20230302039A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
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    • A61K9/127—Liposomes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4712—Cystic fibrosis
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
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    • C12Y—ENZYMES
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    • C12Y306/03049—Channel-conductance-controlling ATPase (3.6.3.49)

Definitions

  • the present invention relates to cystic fibrosis transmembrane regulator (CFTR) mRNA compositions, uses of same, and methods of making and using same.
  • CFTR cystic fibrosis transmembrane regulator
  • Cystic fibrosis is an autosomal inherited disorder resulting from mutation of the
  • CFTR gene which encodes a chloride ion channel believed to be involved in regulation of multiple other ion channels and transport systems in epithelial cells. Loss of function of CFTR results in chronic lung disease, aberrant mucus production, and dramatically reduced life expectancy. See generally Rowe et al, New Engl. J. Med. 352, 1992-2001 (2005).
  • in vitro synthesis of mRNA is generally preferable to synthesis by cells due to the absence of normal cellular mRNA and other cellular components which constitute undesirable contaminants
  • in vitro synthesis of mRNA with a long coding sequence such as CFTR mRNA
  • FFL relatively short coding sequence
  • US2010/0203627 and US2011/0035819 discuss the therapeutic administration of CFTR mRNA but provide neither a demonstrated reduction to practice of production of functional CFTR in the lung following administration of CFTR mRNA or sufficient guidance for overcoming the difficulties associated with inducing CFTR expression in the lung using in vz ' tro-transcribed CFTR mRNA. These include difficulties with achieving in vitro synthesis of the mRNA and difficulties specific to the interactions of mRNA compositions with lung-specific substances that investigators such as Andries et al, supra, have found to render mRNA compositions ineffective Attorney Docket No.: 2006685-0455 for induction of expression even while corresponding DNA-based compositions did provide some level of expression.
  • the present invention is based, in part, on the development of formulations of
  • compositions, methods, and uses according to the invention can provide CFTR expression in the lung of a large mammal with a favorable safety profile suitable for effective treatment of cystic fibrosis.
  • the present invention provides a method of in vivo production of CFTR, in particular, in the lung of a subject (e.g., a mammal) in need of delivery by delivering an mRNA encoding a CFTR protein.
  • the mRNA encoding a CFTR protein is delivered directly to the lung of the subject.
  • a "CFTR protein” encompasses any full length, fragment or portion of a CFTR protein which can be used to substitute for naturally-occurring CFTR protein activity and/or reduce the intensity, severity, and/or frequency of one or more symptoms associated with Cystic fibrosis.
  • a suitable CFTR protein according to the present invention may have an amino acid sequence identical to the wild-type human CFTR protein (SEQ ID NO: 1). In some embodiments, a suitable CFTR protein according to the present invention may have an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the wild-type human CFTR protein (SEQ ID NO: 1).
  • the invention provides a method of inducing CFTR expression in epithelial cells in a lung of a mammal, the method comprising contacting the epithelial cells in the lung of the mammal with a composition, wherein: the composition is a pharmaceutical composition comprising an in vitro transcribed mRNA;the in vitro transcribed mRNA comprises a coding sequence which encodes SEQ ID NO: 1.
  • the Attorney Docket No.: 2006685-0455 in vitro transcribed mRNA comprises a coding sequence which encodes an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.
  • the invention provides a method of inducing CFTR expression in a mammalian target cell, the method comprising contacting the mammalian target cell with a composition, the composition comprising an in vitro transcribed mRNA encoding the amino acid sequence of SEQ ID NO: 1.
  • the in vitro transcribed mRNA comprises a coding sequence which encodes an amino acid sequence at least about 70%>, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.
  • the invention provides a non-naturally occurring mRNA molecule comprising a coding sequence, a 5'-UTR, and a 3'-UTR, wherein the coding sequence encodes the amino acid sequence of SEQ ID NO: 1 and the coding sequence is at least 80% identical to SEQ ID NO: 3.
  • the coding sequence encodes the amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 and/or the coding sequence is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3.
  • the invention provides a non-naturally occurring mRNA molecule comprising a coding sequence, a 5'-UTR, and a 3'-UTR, wherein the coding sequence encodes the amino acid sequence of SEQ ID NO: 1 and the coding sequence comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%o, at least 90%>, or at least 95% of the non-wild-type bases listed in Table 1 at the positions of the coding sequence listed in Table 1 relative to the wild-type coding sequence of SEQ ID NO: 2.
  • the invention provides a non-naturally occurring mRNA molecule comprising a coding sequence, a 5'-UTR, and a 3'-UTR, wherein the coding sequence encodes the amino acid sequence of SEQ ID NO: 1 and the coding sequence comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%), at least 90%, or at least 95% of the non-wild-type bases listed in Table 2 at the
  • the invention provides a non-naturally occurring mRNA molecule comprising a coding sequence for a signal peptide.
  • the invention provides a non-naturally occurring mRNA comprising a coding sequence for a growth hormone leader sequence.
  • the invention provides a non-naturally occurring mRNA comprising a coding sequence of SEQ ID NO: 18 or SEQ ID NO: 19.
  • the invention provides a non-naturally occurring mRNA comprising a coding sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% to SEQ ID NO: 18 or SEQ ID NO: 19.
  • the invention provides a non-naturally occurring mRNA molecule comprising a sequence of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ UD NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.
  • the invention provides a non-naturally occurring mRNA molecule comprising a sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% to any of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ UD NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.
  • the invention provides a polynucleotide comprising a sequence complementary to the sequence of an mRNA according to the invention.
  • the invention provides a composition comprising the polynucleotide according to the invention, an RNA polymerase, and nucleoside triphosphates.
  • the invention provides a pharmaceutical composition comprising an mRNA according to the invention.
  • the invention provides a nebulization or aerosolization apparatus loaded with a pharmaceutical composition according to the invention.
  • the invention provides a cultured cell comprising an mRNA according to the invention and functional CFTR expressed from the mRNA.
  • the invention provides a use of a pharmaceutical composition according to the invention for the induction of expression of functional CFTR.
  • the invention provides a method of inducing CFTR
  • the method comprising contacting the Attorney Docket No.: 2006685-0455 epithelial cells with a composition, wherein the composition is a pharmaceutical composition comprising an mRNA according to the invention.
  • the invention provides a method of inducing CFTR expression in a mammalian target cell, the method comprising contacting the mammalian target cell with a composition, the composition comprising an mRNA according to the invention.
  • the present invention provides a method of treating cystic fibrosis by administering to the lung of a subject in need of treatment an mRNA comprising a coding sequence which encodes SEQ ID NO: l .
  • the present invention provides a method of treating cystic fibrosis by administering to the lung of a subject in need of treatment an mRNA comprising a coding sequence which encodes an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the wild-type human CFTR protein (SEQ ID NO: 1).
  • the present invention provides a method of treating cystic fibrosis by administering to the lung of a subject in need of treatment an mRNA comprising a coding sequence of SEQ ID NO:3. In some embodiments, the present invention provides a method of treating cystic fibrosis by
  • an mRNA comprising a coding sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3.
  • the present invention provides methods for making an mRNA encoding a CFTR protein as described herein.
  • the invention provides a method of making CFTR mRNA in vitro, comprising contacting an isolated polynucleotide with an RNA polymerase in the presence of nucleoside triphosphates, wherein: the isolated polynucleotide and RNA polymerase are not contained within a cell; the isolated nnlvrmd p ntirl p i « a template for the RNA polymerase; the isolated polynucleotide comprises a Attorney Docket No.: 2006685-0455 promoter operably linked to a template sequence; the template sequence comprises a coding sequence complement which is complementary to a sequence encoding SEQ ID NO: 1; and: (a) the template sequence comprises fewer cryptic promoters than the complement of SEQ ID NO: 2, (b) the template sequence comprises fewer direct and/or inverted repeats
  • the invention provides a method of making CFTR mRNA in vitro, comprising contacting an isolated polynucleotide according to the invention with an RNA polymerase in the presence of nucleoside triphosphates, wherein: the isolated
  • polynucleotide and RNA polymerase are not contained within a cell; the isolated polynucleotide is a template for the RNA polymerase; and the isolated polynucleotide comprises a promoter operably linked to a template sequence, and the RNA polymerase synthesizes mRNA comprising a coding sequence encoding SEQ ID NO: 1.
  • the in vitro transcribed mRNA is a naturally occurring or wild-type mRNA encoding human CFTR (SEQ ID NO: 2) modified to include non-naturally occurring UTRs.
  • the in vitro transcribed mRNA is a non-naturally occurring mRNA as described above.
  • Figure 1A Detection of mature "C" band for human CFTR protein 24 hours after transfection with human CFTR mRNA. Successful protein production was observed for both unmodified and modified (SNIM) mRNA (comprising 25% of 2-thiouridine and 5- methylcytidine). Immunoprecipitation was performed using R&D Systems MAB25031 antibody and detection using Ab570.
  • Figure IB Western Blot analysis of CFTR KO mouse lungs 24 hour postexposure of PEI/unmodified human CFTR mRNA nanoparticles. Mice were treated via nebulization (Pari Boy jet nebulizer) over the course of approximately one hour.
  • FIG. 1 Current-voltage plot of 8-Br-cAMP evoked currents of treated (4 ug hCFTR mRNA) and untreated HEK293T cells. A large current is induced within the hCFTR mRNA transfected cells as compared to the untreated cells. Treated cells that were exposed to a specific CFTR protein inhibitor, CFTRinh-172, show a marked reduction (-89%) in CI- ion current flow.
  • FIG. 3 Histogram plots of 8-Br-cAMP evoked currents of treated (4 ug hCFTR mRNA) and untreated HEK293T cells upon application of a +80mV membrane potential. A large current is induced within the hCFTR mRNA transfected cells as compared to the untreated cells. Treated cells that were exposed to a specific CFTR protein inhibitor, CFTRinh-172, show a marked reduction (-89%) in CI- ion current flow.
  • Figure 4 Current-voltage plot comparing profiles of HEK 293 cells of native, forskolin and GlyH-101 exposure. No significant changes in current were observed in any scenario.
  • Figure 5 Current- voltage plot of forskolin-evoked currents of treated (4 ug hCFTR mRNA) and untreated HEK293 cells. A large current is induced within the hCFTR Attorney Docket No.: 2006685-0455 mRNA transfected cells as compared to the untreated cells. Treated cells that were exposed to a specific CFTR protein inhibitor, GlyH-101, show a marked reduction (-95%) in CI- ion current flow as demonstrated in the step plot (+100 mV) on the right side of the graph.
  • Figure 7 In situ hybridization of human CFTR mRNA treated CFTR KO mouse lungs at different magnification views (up to 20x magnification). Mice were exposed to 30 ug of encapsulated unmodified hCFTR mRNA in PEI nanoparticles via intratracheal administration.
  • Figure 8 High magnification (40x) representative lung section demonstrating in situ hybridization of human CFTR mRNA treated (right) CFTR KO mouse lungs. Human CFTR mRNA was detected in the apical cytoplasm of target bronchial epithelial cells 24 hours post administration. Mice were exposed to 30 ug of encapsulated unmodified hCFTR mRNA in PEI nanoparticles via intratracheal administration.
  • Figure 9 Comparison of in situ hybridization staining of human CFTR mRNA treated CFTR KO mouse lungs at six hours (left) and 24 hours (right) post-administration. Mice were exposed to 30 ug of encapsulated unmodified hCFTR mRNA in PEI nanoparticles via intratracheal administration. Intense positive intracellular staining is observed within six hours throughout both lungs within bronchial and alveolar regions while substantial positive staining is still observed at 24 hours post-administration.
  • Figure 10 In situ hybridization of human CFTR mRNA in untreated (PBS) (top) and treated (bottom) CFTR KO mouse lungs. Mice were exposed to 15 ug of encapsulated unmodified hCFTR mRNA in CI 2-200 lipid nanoparticles via intratracheal administration. Substantial positive staining is observed throughout both lungs at 6 hours post-administration.
  • FIG. 11 High magnification (40x) representative lung sections demonstrating in situ hybridization of human CFTR mRNA treated CFTR KO mouse lungs. Human CFTR mRNA was detected in the apical cytoplasm of target bronchial epithelial (left) as well as intracellular alveolar regions (right) six hours post administration. Mice were exposed to 15 ug Attorney Docket No.: 2006685-0455 of encapsulated unmodified hCFTR mRNA in CI 2-200 lipid nanoparticles via intratracheal administration.
  • Mouse anti-human CFTR MAI -935 (Chemicon): (B) - Mouse anti-human CFTR AB570 (Cystic Fibrosis Foundation): (C) - Mouse anti-human CFTR AB596 (Cystic Fibrosis Foundation): (D) Rabbit anti-human CFTR G449 (Rockefeller University). Arrow indicates CFTR.
  • Lane 1 T84 cells (positive control)
  • Lane 2 untreated pig lung tissue (300 mg)
  • Lane 3 treated pig lung tissue (697 mg)
  • Lane 4 treated pig lung tissue (163 mg).
  • Figure 15 Immunoprecipitation and Western blotting of a mouse at 24 hrs post
  • T84 cells served a positive control showing the mature glycosylated C-band and the mannosylated B-band of hCFTR. "supernatant” remaining cellular extract fraction without immunoprecipitated fraction. "IP” immunoprecipitated fraction.
  • Figure 16 Immunoprecipitation of hCFTR from T84 cells using MAB25031 followed by immunodetection using AB570 (A) and MAB1660 (B).
  • Figure 17 Immunoprecipitation of CFTR from NIH3T3 cells at 72 hrs post transfection with different constructs.
  • Figure 18 Immunoprecipitation of CFTR from NIH3T3 cells at 72 hrs post transfection with different constructs using 500 ug protein and MAB1660 (left and center panels) and increased amount of total protein (8 mg) using MAB25031 (right panel).
  • Lane 1 sample from luciferase-negative left caudal lobe of pig #2
  • Lane 2 sample from luciferase-positive lung regions of pig #1.
  • the nebulizer was connected in-line to the ventilation system (right, see white arrow).
  • Figure 21 Luciferase expression measured in homogenates of pig tissue specimens from different lung regions after aerosol administration of 1 mg FFL SNIM RNA in the PEI Formulation of Example 6 with the EFlow mesh nebulizer. Lung specimens were ex vivo cultured overnight before luciferase measurements (pg luciferase/mg lung tissue).
  • Figure 22 BLI of luciferase expression in representative pig tissue specimens from different lung regions after aerosol administration of 1 mg FFL SNIM RNA in the PEI Formulation of Example 6. Lung specimens were ex vivo cultured overnight before
  • FIG. 23 BLI imaging of luciferase expression in representative pig tissue specimens from different lung regions after aerosol administration of 1 mg FFL SNIM RNA in the PEI Formulation of Example 6 using a PARI BOY jet nebulizer. Lung specimens were ex vivo cultured overnight before measurements.
  • Figure 24 BLI of luciferase expression in representative pig tissue specimens from different lung regions after aerosol administration of each 1 mg FFL SNIM RNA and hCFTR mRNA in the PEI Formulation of Example 6 using an Aeroneb mesh nebulizer. Lung specimens were ex vivo cultured overnight before measurements.
  • Figure 25 BLI of luciferase expression in representative pig tissue specimens from different lung regions after aerosol administration of 1 mg FFL SNIM RNA in "SHIRE Formulation #3" (HGT5001 :DOPE:Chol:DMGPEG2K (50:25:20:5) (mol ratio) using an Aeroneb mesh nebulizer. Lung specimens were ex vivo cultured overnight before
  • Figure 26 BLI of luciferase expression in pig tissue specimens from different lung regions from one untreated control pig. The other untreated control pig showed the same result (data not shown).
  • FIG. 27 BLI of luciferase expression in lung specimens of once-treated pigs #3 and #6. Aerosol administration of each 1 mg FFL SNIM RNA and hCFTR SNIM RNA in the PEI Formulation of Example 6 was performed using an Aeroneb mesh nebulizer. Slices of the entire pig lung are shown. Upper three rows: pig #3, lower three rows: pig #6.
  • Figure 28 BLI of luciferase expression in lung specimens of twice-treated pigs
  • Example 6 Aerosol administration of each 1 mg FFL SNIM RNA and hCFTR SNIM RNA in the PEI Formulation of Example 6 was performed using an Aeroneb mesh nebulizer. Slices of the entire pig lung are shown. Upper three rows: pig #4, lower three rows: pig #8.
  • FIG. 29 BLI of luciferase expression in lung specimens of three times-treated pigs #1 and #2. Aerosol administration of each 1 mg FFL SNIM RNA and hCFTR-mRNA SNIM RNA in the PEI Formulation of Example 6 were performed using an Aeroneb mesh nebulizer. Slices of the entire pig lung are shown. Upper three rows: pig #1, lower three rows: pig #2.
  • FIG 30 Luciferase IHC on lung tissue of three times treated pig #1. Aerosol administration of each 1 mg FFL SNIM RNA and hCFTR SNIM RNA in the PEI Formulation of Example 6 was performed using an Aeroneb mesh nebulizer. Luciferase expression appeared in reddish-pink colour (Anti-Luciferase pAb 1 :300, G7451, Promega, Refine AP-Kit, chromogen: New fuchsine).
  • FIG. 31 Highly BLI-positive lung tissue of threefold treated pig #1 was subjected to hCFTR IP/WB.
  • Lane 2 untreated pig lung tissue (300 mg)
  • Lane 3 treated pig lung tissue (697 mg)
  • Lane 4 treated pig lung tissue (163 mg).
  • Mature complex-glycosylated hCFTR appeared as the disperse so-called C-band.
  • Mannose- rich hCFTR appeared as the more dense so-called B-band.
  • hCFTR expression was observed in T84 cells and pig lung tissue of hCFTR SNIM RNA treated pig #1, whereas no hCFTR expression was observed in untreated pigs.
  • FIG. 32 Immunoprecipitation of hCFTR using MAB25031 and subsequent immunodetection using AB570 from pig lung samples post hCFTR SNIM RNA delivery in the PEI Formulation of Example 6.
  • Lane 1 sample from luciferase-negative left caudal lobe of pig #2
  • Lane 2 sample from luciferase-positive lung regions of pig #1.
  • Figure 33 A&B In vitro transfection of HEK 293T cells with C-terminal Hisio tagged (CO-CFTR-C-Hisio) and non-tagged (CO-CFTR) codon optimized human CFTR SNIM RNA. Following transfection, whole cell lysate was collected and analyzed for human CFTR expression by Western blot using (A) anti-CFTR antibody #217 and (B) anti-His antibody 1187. Transfected samples were compared to non-transfection HEK 293T control lysate (Lane 3).
  • Figure 33 C In vitro transfection of HEK 293T cells with SNIM RNA encoding codon optimized human CFTR with a growth hormone leader sequence and a (GH-CO-CFTR) or SNIM RNA encoding a C-terminal Hisio tagged codon optimized human CFTR (CO-CFTR- C-Hisio). Following transfection, whole cell lysate was collected and analyzed for human CFTR expression by Western blot using anti-CFTR antibody #217. Transfected samples were compared to non-transfection HEK 293T control lysate (Lane 3).
  • FIG. 34 In vivo transfection of CFTR knockout mice with C-terminal Hisio tagged codon optimized human CFTR SNIM RNA encapsulated within either a lipid (cKK-E12) or polymeric (PEI) nanoparticle formulation. Following nebulized delivery of each respective mRNA formulation, Right and Left lung tissue lysate was collected and analyzed for CFTR expression by Western blot using anti-His antibody 1187. Control CFTR knockout lung tissue and CFTR-Hisio HEK293 lysate was used as a negative and positive controls respectively.
  • Figure 35 Bio luminescent detection of FFL expression in porcine lung samples collected following nebulization with water for injection.
  • Figure 36 Bio luminescent detection of FFL expression in porcine lung samples collected following nebulization with 1 mg FFL SNIM RNA + 1 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.
  • Figure 37 Bio luminescent detection of FFL expression in porcine lung samples collected following nebulization with 1 mg FFL SNIM RNA + 5 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.
  • FIG. 38 Bio luminescent detection of FFL expression in porcine lung samples collected following nebulization with 1 mg FFL SNIM RNA + 10 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.
  • Figure 39 Relative quantification of CFTR expression in different chorots.
  • Figure 40 Representative example of a "CFTR-positive" bronchi with at least one epithelial cell detected within the epithelial cell layer and displaying a clear membrane localized CFTR signal via CFTR immunohistochemical staining using an anti-CFTR antibody.
  • FIG 41 Immunohistochemical staining of CFTR in porcine lung following aerosol delivery of control (WFI) or 5 mg CO-CFTR SNIM RNA.
  • Figure 42 Represents a "low" CFTR expression level, assayed in porcine lung by immunohistochemical staining with anti-CFTR following aerosol delivery of 5 mg CO-CFTR SNIM RNA.
  • Figure 43 Represents a "medium" CFTR expression level, assayed in porcine lung by immunohistochemical staining with anti-CFTR following aerosol delivery of 5 mg CO- CFTR SNIM RNA.
  • Figure 44 Represents a "high" CFTR expression level, assayed in porcine lung by immunohistochemical staining with anti-CFTR following aerosol delivery of 5 mg CO-CFTR SNIM RNA.
  • FIG. 45 Immunohistochemical staining of CFTR in porcine lung following aerosol delivery of control (WFI) or 10 mg CO-CFTR SNIM RNA.
  • Figure 47 Illustrates multiplex nucleic acid in situ detection of (A) ubiquitin C and (B) dap B in porcine lung, post aerosol delivery of water for injection by nebulizer.
  • Figure 48 Illustrates multiplex nucleic acid in situ detection of (A) ubiquitin C and (B) dap B in porcine lung, post aerosol delivery of 1 mg FFL SNIM RNA + 10 mg CO- CFTR SNIM RNA in a branched 25 kDa PEI formulation.
  • Figure 49 Illustrates multiplex nucleic acid in situ detection of (A) right cranialis and (B) left cranialis in porcine, post aerosol delivery of water for injection by nebulizer.
  • Figure 50 Illustrates multiplex nucleic acid in situ detection of (A) right cranialis and (B) left cranialis in porcine, post aerosol delivery of 1 mg FFL SNIM RNA + 1 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.
  • Figure 51 Illustrates multiplex nucleic acid in situ detection of (A) right cranialis and (B) left cranialis in porcine, post aerosol delivery of 1 mg FFL SNIM RNA + 5 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.
  • Figure 52 Illustrates multiplex nucleic acid in situ detection of (A) right cranialis and (B) left cranialis in porcine, post aerosol delivery of 1 mg FFL SNIM RNA + 10 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.
  • FIG. 53 Illustrates positive detection of active firefly luciferase (FFL) protein in a treated pig lung via luminescence upon exposure to FFL/CO-CFTR-C-HislO mRNA encapsulated cK -E12 lipid nanoparticles.
  • Pigs were treated with 1 mg FFL + 9 mg CO-CFTR- C-HislO mRNA encapsulated lipid nanoparticles via nebulization using a Pari jet nebulizer and sacrificed 24 hours post-treatment.
  • FFL luminescence was visualized using an IVIS
  • Figure 55 Illustrates exemplary results of hCFTR expression in HEK cells transfected using neubilized complexes given to pigs 13, 14 and 15 (5 mg dose) and in HEK cells transfected using neubilized complexes given to pigs 19, 20 and 21 (10 mg dose).
  • Figure 56 Illustrates exemplary results of hCFTR expression in HEK cells transfected using neubilized complexes given to pig 16 (5 mg dose), 22 (10 mg dose) and 67 (1 mg dose).
  • Figure 57 Illustrates exemplary results of hCFTR expression in HEK cells transfected using neubilized complexes given to pigs 17, 18 (5 mg dose), 23, 24 (10 mg dose) Attorney Docket No.: 2006685-0455
  • polynucleotide is generally used to refer to a nucleic acid (e.g., DNA or RNA).
  • the terms polynucleotide, nucleic acid, DNA, RNA, and mRNA include such molecules that are comprised of: standard or unmodified residues; nonstandard or modified residues; and mixtures of standard and nonstandard residues.
  • mRNA is used to refer to modified and unmodified
  • RNA including both a coding region and a noncoding region.
  • coding region of an mRNA generally refers to that portion that when translated results in the production of an expression product, such as a polypeptide, protein, or enzyme.
  • a "nonstandard nucleobase” is a base moiety other than the natural bases adenine
  • the nonstandard nucleobase is an analog of a specific nucleobase (A, C, G, T, or U) when its base pairing properties in a nucleic acid double helix and locus of incorporation by DNA or RNA polymerases in a nucleic acid double helix (including a local RNA-DNA helix such as that formed during transcription of a DNA template by RNA polymerase) are most similar to one of the five previously listed nucleobases, with the exception that analogs of T will generally also be analogs of U and vice versa.
  • an analog of a base is not a mismatch to the natural base; for example, pseudouridine matches uridine, 5-methylcytidine matches cytidine, etc.
  • GC content is the fraction or percentage of total nucleobase residues in a nucleic acid sequence that are guanine residues, cytosine residues, or analogs thereof.
  • a 100 nt sequence that contains exactly 30 cytosines, exactly 30 guanines, exactly one cytosine analog, and exactly one guanine analog has a GC richness of 62%.
  • a "disfavored codon” refers to a codon which is translated less efficiently or rapidly by mammalian cells than another codon for the same amino acid residue. Disfavored codons generally include codons with an A or U in the 3rd or "wobble" position of the codon. For a discussion of disfavored codons, see, e.g., U.S. Patent Publication
  • a "non-naturally occurring mRNA molecule” is an mRNA that is not produced through normal transcription and splicing processes of wild-type cells.
  • An mRNA may qualify as non-naturally occurring by virtue of its sequence (e.g., a series of codons and/or one or more UTRs that do not present in any naturally-occurring CFTR mRNA) and/or because it includes nonstandard nucleotide residues.
  • a non-naturally occurring mRNA molecule may be in vitro synthesized.
  • the NWT column indicates the non- wild-type base at the position (Pos.) in the CFTR coding sequence (see, e.g., SEQ ID NO: 3), and the WT column indicates the wild-type base at the same position (see, e.g., SEQ ID NO: 2 or the RefSeq entry for human CFTR (accession no. NM 000492.3, Feb. 10, 2013, version, available from GenBank; note that the sequence of NM 000492.3 contains noncoding sequence such that the coding sequence occurs at position 133 to 4575, such that, for example, position 7 in the tables below corresponds to position 139 of the NM 000492.3 sequence).
  • the invention in addition to providing methods of producing functional CFTR in vivo using naturally occurring or wild-type CFTR mRNA (and compositions comprising that mRNA), the invention also provides non-naturally occurring CFTR mRNA that encodes CFTR protein (e.g., SEQ ID NO:l). In some embodiments, the non-naturally occurring CFTR mRNA is purified or isolated.
  • the non-naturally occurring CFTR mRNA is present in a cell.
  • the cell comprising the non-naturally occurring CFTR mRNA did not synthesize the non-naturally occurring CFTR mRNA and/or does not comprise DNA Attorney Docket No.: 2006685-0455 complementary to the non-naturally occurring CFTR mRNA and/or a functional CFTR gene; the cell may optionally comprise an inactive CFTR gene, such as a CFTR gene with a nonsense, missense, frameshift, insertion, or deletion mutation that renders the expression product of the gene nonfunctional.
  • the cell comprising the non-naturally occurring CFTR mRNA further comprises functional CFTR protein translated from the non-naturally occurring CFTR mRNA.
  • the cell may be, e.g., a lung epithelial cell, a liver cell, or a kidney cell. In some embodiments, the cell is in a cell culture.
  • CFTR mRNA according to the invention comprises a coding sequence with fewer complements of cryptic promoters than SEQ ID NO: 2 (i.e., the coding sequence of wild-type human CFTR), fewer direct and/or inverted repeats than SEQ ID NO: 2, fewer disfavored codons than SEQ ID NO: 2, and/or the GC content of the coding sequence is lower than the GC content of SEQ ID NO: 2.
  • Cryptic promoters, direct and/or inverted repeats and/or disfavored codons of a sequence may be recgnized by one skilled in the art using routine methods.
  • the direct and/or inverted repeat content of a sequence can be determined by sequence analysis (Liu et al, Journal of Theoretical Biology (2014) 344: 19-30).
  • the cryptic promoter content of a sequence can also be determined by sequence analysis, e.g., presence of Shine-Dalgarno sequences within construct or the like.
  • CFTR mRNA according to the invention is in vitro- transcribed, i.e., the mRNA was synthesized in an artificial setting not within a biological cell (e.g., a cell free in vitro transcription system).
  • in vitro transcription involves providing a DNA template comprising a promoter and a sequence complementary to the desired mRNA (which may be circular or linear), an RNA polymerase, and nucleoside triphosphates in suitable reaction conditions (salts, buffers, and temperature). RNase inhibitors, reducing agents, and/or pyrophosphatase may be present in the reaction mixture.
  • the RNA polymerase is T7 RNA polymerase.
  • the CFTR mRNA according to the invention comprises a coding sequence comprising at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, Attorney Docket No.: 2006685-0455 at least 75%, at least 80%>, at least 85%, at least 90%>, or at least 95% of the non-wild-type bases listed in Table 1 at the positions of the coding sequence listed in Table 1 relative to the wild- type coding sequence of SEQ ID NO: 2.
  • the CFTR mRNA according to the invention comprises a coding sequence comprising at least 50%, at least 55%, at least 60%>, at least 65%, at least 70%>, at least 75%, at least 80%>, at least 85%, at least 90%, or at least 95% of the non-wild-type bases listed in Table 2 at the corresponding positions of the coding sequence listed in Table 2 relative to the wild-type coding sequence of SEQ ID NO: 2.
  • the present invention comprises a non-naturally occurring
  • CFTR mRNA comprising a coding sequence of SEQ ID NO: 3. Additional exemplary non- naturally occurring CFTR mRNA coding sequences are described in the Brief Description of Sequences section, such as, for example, SEQ ID NOs:9, 10, 11, 12, 13, 14, 15, 16, or 17.
  • the present invention provides a CFTR mRNA comprising a coding sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 3, 9, 10, 11, 12, 13, 14, 15, 16, or 17.
  • a non-naturally occurring CFTR mRNA comprises a 5'UTR, 3'UTR, a signal peptide coding sequence or a cap or tail structure as described below.
  • CFTR mRNAs comprising coding sequence which differs from wild-type CFTR coding sequence can provide advantages with respect to efficacy and ease of preparation.
  • in vitro transcription reactions using a polynucleotide comprising template sequence complementary to the CFTR coding sequence can give greater RNA yield; a polynucleotide comprising said template sequence can be more stable (i.e., less prone to mutation) during growth in a host cell, reducing the amount of purification needed to generate template usable in a reaction; and the in vivo translation of an mRNA comprising the coding sequence can be higher.
  • an mRNA encoding a CFTR protein incorporates a nucleotide sequence encoding a signal peptide.
  • signal peptide refers to a peptide present at a newly synthesized protein that can target the protein towards the secretory pathway.
  • the signal peptide is cleaved after translocation into the endoplasmic reticulum following translation of the mRNA.
  • Signal peptide is also referred to as signal sequence, leader sequence or leader peptide.
  • a signal peptide is a short (e.g.,
  • a signal peptide may be present at the
  • the incorporation of a signal peptide encoding sequence on a CFTR encoding mRNA may facilitate the secretion and/or production of the CFTR protein in vivo.
  • a suitable signal peptide for the present invention can be a heterogeneous sequence derived from various eukaryotic and prokaryotic proteins, in particular secreted proteins.
  • a suitable signal peptide is a leucine-rich sequence. See Yamamoto Y et al. (1989), Biochemistry, 28:2728-2732, which is incorporated herein by reference.
  • a suitable signal peptide may be derived from a human growth hormone (hGH), serum albumin preproprotein, Ig kappa light chain precursor, Azurocidin preproprotein, cystatin- S precursor, trypsinogen 2 precursor, potassium channel blocker, alpha conotoxin lpl .3, alpha conotoxin, alfa-galactosidase, cellulose, aspartic proteinase nepenthesin-1, acid chitinase, K28 prepro-toxin, killer toxin zygocin precursor, and Cholera toxin.
  • Exemplary signal peptide sequences are described in Kober, et al, Biotechnol. Bioeng., 110: 1164-73, 2012, which is incorporated herein by reference.
  • a CFTR encoding mRNA may incorporate a sequence encoding a signal peptide derived from human growth hormone (hGH), or a fragment thereof.
  • hGH human growth hormone
  • a non-limiting nucleotide sequence encoding a hGH signal peptide is show below.
  • hGH human growth hormone sequence
  • an mRNA according to the present invention may incorporate a signal peptide encoding sequence having at least 50%, 55%, 60%>, 65%, 70%, 75%,
  • the mRNA comprises a sequence in its 3'-UTR which is identical to SEQ ID NO: 5 or is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 5.
  • the mRNA comprises a poly-A tail.
  • the poly-A tail has a length of at least 70, 100, 120, 150, 200, 250, 300, 400, or 500 residues. In some embodiments, the poly-A tail has a length ranging from 70 to 100, 100 to 120, 120 to 150, 150 to 200, or 200 to 300, 300 to 400, or 400 to 500 residues.
  • Poly A tails can be added using a variety of art-recognized techniques. For example, long poly A tails can be added to synthetic or in vitro transcribed RNA using poly A polymerase (Yokoe, el al. Nature Biotechnology. 1996; 14: 1252-1256). A transcription vector can also encode long poly A tails. In addition, poly A tails can be added by transcription directly from PCR products.
  • Poly A may also be ligated to the 3' end of a sense RNA with RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).
  • a poly-U or poly-C tail may be used instead or in addition to a poly-A tail.
  • CFTR encoding mRNAs may include a 3' poly(C) tail structure.
  • a suitable poly-C tail on the 3' terminus of mRNA typically include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides).
  • the poly-C tail may be added to the poly-A tail or may substitute the poly-A tail.
  • the mRNA comprises a 5'-cap, for example, a capl structure.
  • a 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5 '5 '5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase.
  • GTP guanosine triphosphate
  • cap structures include, but are not limited to, m7G(5')ppp (5 * (A,G(5 * )ppp(5 * )A and G(5 * )ppp(5 * )G.
  • the mRNA comprises one or more nonstandard nucleotide residues.
  • the nonstandard nucleotide residues may include, e.g., 5-methyl-cytidine ("5mC"), pseudouridine (“ ⁇
  • mRNA may be SNIM RNA.
  • SNIM RNA is an acronym of Stabilized Non-Immunogenic Messenger RNA, designating messenger RNAs produced by in vitro transcription (IVT) including certain percentages of modified nucleotides in the IVT reaction as described in PCT Publication WO 2011/012316.
  • IVT in vitro transcription
  • SNIM RNA used in the Examples disclosed herein was produced by IVT in which 25% of U residues were 2-thio-uridine and 25% of C residues were 5-methylcytidine.
  • the presence of nonstandard nucleotide residues may render an mRNA more stable and/or less immunogenic than a control mRNA with the same sequence but containing only standard residues.
  • the mRNA may comprise one or more nonstandard nucleotide residues chosen from isocytosine,
  • Certain embodiments may further include additional modifications to the furanose ring or nucleobase. Additional modifications may include, for example, sugar modifications or substitutions (e.g., one or more of a 2'-0-alkyl modification, a locked nucleic acid (LNA)).
  • LNA locked nucleic acid
  • the R As may be complexed or hybridized with additional polynucleotides and/or peptide polynucleotides (PNA).
  • PNA polynucleotides
  • such modification may include, but are not limited to a 2'-deoxy-2'-fluoro modification, a 2'-0-methyl
  • any of these modifications may be present in 0-100% of the nucleotides— for example, more than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of the constituent nucleotides individually or in combination.
  • compositions comprising CFTR mRNA
  • the mRNA molecules of the invention may be administered as naked or unpackaged mRNA.
  • the administration of the mRNA in the compositions of the invention may be facilitated by inclusion of a suitable carrier.
  • the carrier is selected based upon its ability to facilitate the transfection of a target cell with one or more mRNAs.
  • carrier includes any of the standard pharmaceutical carriers, vehicles, diluents, excipients and the like which are generally intended for use in connection with the administration of biologically active agents, including mRNA.
  • the carriers employed in the compositions of the invention may comprise a liposomal vesicle, or other means to facilitate the transfer of a mRNA to target cells and/or tissues.
  • Suitable carriers include, but are not limited to, polymer based carriers, such as polyethyleneimine (PEI) and multi-domain-block polymers, lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphor-silicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, dry powders, poly(D-arginine), nanodendrimers, starch-based delivery systems, micelles, emulsions, sol-gels, niosomes, plasmids, viruses, calcium
  • PEI polyethylenei
  • the carrier comprises an organic cation, such as a cationic lipid or a cationic organic polymer. If present, the cationic lipid may be a component of liposomal vesicles encapsulating the mR A.
  • the carrier is formulated using a polymer as a carrier, alone or in combination with other carriers.
  • Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL and polyethylenimine (PEI).
  • PEI When PEI is present, it may be branched PEI of a molecular weight ranging from 10 to 40 kDa, e.g., 25 kDa branched PEI (Sigma #408727). Additional exemplary polymers suitable for the present invention include those described in PCT Publication WO2013182683, the contents of which is hereby incorporated by reference.
  • liposomal carriers to facilitate the delivery of polynucleotides to target cells is contemplated by the present invention.
  • Liposomes e.g., liposomal lipid nanoparticles
  • Liposomes are generally useful in a variety of applications in research, industry, and medicine, particularly for their use as carriers of diagnostic or therapeutic compounds in vivo (Lasic, Trends).
  • Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that mmnrisfi snatiallv separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of the liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.).
  • the mRNA is complexed with lipid nanoparticles to facilitate delivery to the target cell.
  • the compositions of the invention may be combined with a multi-component lipid mixture employing one or more cationic lipids, additional lipids such as non-cationic lipids (also referred to as helper lipids), cholesterol-based lipids, and/or PEGylated lipids for mRNA encapsulation.
  • a suitable lipid nanoparticle contains a cationic lipid.
  • cationic lipid refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH.
  • Some cationic lipids in particular, those known as titratable or pH-titratable cationic lipids are particularly effective in delivering mRNA.
  • Several cationic (e.g., titratable) lipids have been described in the literature, many of which are commercially available. Particularly suitable cationic lipids for use in the
  • compositions and methods of the invention include those described in international patent publications WO 2010/053572 (and particularly, C12-200 described at paragraph [00225]) and
  • the cationic lipid cK -E12 is used (disclosed in WO 2013/063468), the teachings of which are incorporated herein by reference in their entirety.
  • DOTMA (2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
  • DOTMA can be formulated alone or can be combined with the neutral lipid, dioleoylphosphatidyl-ethanolamine or "DOPE" or other cationic or non-cationic lipids into a liposomal transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of nucleic acids into target cells.
  • DOPE dioleoylphosphatidyl-ethanolamine
  • Other suitable cationic lipids include, for example, 5-
  • ycinedioctadecylamide or "DOGS” 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminium or "DOSPA" (Behr et al. Proc. Nat. 7 Acad. Sci. 86, 6982 (1989); U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761), l,2-Dioleoyl-3- Dimethylammonium-Propane or "DODAP”, l,2-Dioleoyl-3-Trimethylammonium-Propane or "DOTAP".
  • Contemplated cationic lipids also include l,2-distearyloxy-N,N-dimethyl-3- aminopropane or "DSDMA", l,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA", 1 ,2- dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA", l,2-dilinolenyloxy-N,N-dimethyl- 3-aminopropane or "DLenDMA", N-dioleyl-N,N-dimethylammonium chloride or "DODAC", N,N-distearyl-N,N-dimethylarnrnonium bromide or "DDAB", N-(l,2-dimyristyloxyprop-3-yl)- N,N-dimethyl-N-hydroxyethyl ammonium bromide or "DMRIE", 3-dimethylamino
  • compositions and methods of the invention employ a lipid nanoparticles comprising an ionizable cationic lipid described in U.S. provisional patent application 61/617,468, filed March 29, 2013 (incorporated herein by reference), such as, e.g,
  • one or more of the cationic lipids present in such a composition comprise at least one of an imidazole, dialkylamino, or guanidinium moiety. In a preferred embodiment, one or more of the cationic lipids does not comprise a quaternary amine.
  • Non- cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC),
  • DSPC distearoylphosphatidylcholine
  • DOPC dioleoylphosphatidylcholine
  • DPPC dipalmitoylphosphatidylcholine
  • dioleoylphosphatidylethanolamine DOPE
  • palmitoyloleoylphosphatidylcholine POPC
  • palmitoyloleoyl-phosphatidylethanolamine POPE
  • dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-l-carboxylate DOPE-mal
  • dipalmitoyl phosphatidyl ethanolamine DPPE
  • dimyristoylphosphoethanolamine DMPE
  • distearoyl-phosphatidyl- ethanolamine DSPE
  • 16-O-monomethyl PE 16-O-dimethyl PE
  • 18-1 -trans PE l-stearoyl-2- oleoyl-phosphatidyethanolamine
  • SOPE l-stearoyl-2- oleoyl-phosphatidyethanolamine
  • a suitable lipid nanoparticle comprises one or more cholesterol-based lipids.
  • suitable cholesterol-based cationic lipids include, for example, cholesterol, PEGylated cholesterol, DC-Choi (N,N-dimethyl-N- ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE.
  • a suitable lipid nanoparticle comprises one or more
  • PEGylated lipid DSPE-PEG-Maleimide-Lectin may be used.
  • Other contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length.
  • the addition of PEGylated lipids may prevent complex aggregation and increase circulation lifetime to facilitate the delivery of the lipsome encapsulated mRNA to the target cell.
  • the composition comprises one of the following combinations of lipids:
  • DODAP DOPE
  • cholesterol DMG-PEG2K
  • HGT5000 DOPE, cholesterol, DMG-PEG2K
  • HGT5001 DOPE, cholesterol, DMG-PEG2K;
  • lipid:mR A ratios can be 5: 1 (mg:mg), 6: 1, 7: 1, 8: 1, 9: 1,
  • N/P ratios can be in the range of 1.1 : 1 up to 10:1 or higher.
  • Example lipid ratios are 40:30:20: 10, 55:20:20:5, 50:25:20:5 (cationic lipid:helper lipid:chol:PEG lipid).
  • the pharmaceutical compositions according to the invention do not comprise a mucolytic agent (e.g., N-acetylcysteine, erdosteine, bromheksin, carbocysteine, guiafenesin, or iodinated glycerol).
  • a mucolytic agent e.g., N-acetylcysteine, erdosteine, bromheksin, carbocysteine, guiafenesin, or iodinated glycerol.
  • a pharmaceutical composition according to the invention such as a cationic lipid-based or PEI-based composition comprising a non-naturally occurring CFTR mR A
  • the apparatus can be, e.g., an instillation, aerosolization, or nebulization apparatus.
  • Suitable apparatuses include, for example, a PARI Boy jet nebulizer, Aeroneb® Lab nebulizer, MicroSprayer®, or EFlow mesh nebulizer.
  • dry powder inhalers or aerosolization apparatuses such as portable inhalers may be used.
  • the present invention provides methods for in vivo production of a CFTR protein, in particular, in a lung of a mammal.
  • the invention provides methods of inducing CFTR expression in epithelial cells in a lung of a mammal, comprising contacting the epithelial cells with a pharmaceutical composition comprising an in vitro transcribed mRNA, wherein the in vitro transcribed mRNA comprises a coding sequence encoding SEQ ID NO: 1 (the amino acid sequence of wild-type human CFTR).
  • the invention also provides uses of pharmaceutical compositions comprising an in vitro transcribed mRNA, wherein the in vitro transcribed mRNA comprises a coding sequence encoding SEQ ID NO: 1, for the induction of CFTR expression in epithelial cells in a lung of a mammal.
  • the invention further provides methods of inducing CFTR expression in a mammalian target cell, the method comprising contacting the mammalian target cell with a composition, the composition comprising an in vitro transcribed mRNA encoding the amino acid sequence of SEQ ID NO: 1.
  • the invention further provides a use of composition, the composition comprising an in vitro transcribed mRNA encoding the amino acid sequence of SEQ ID NO: 1, for the induction of CFTR expression in a mammalian target cell.
  • the in vitro transcribed mRNA is a naturally occurring or wild-type mRNA encoding human CFTR (SEQ ID NO: 2). In other embodiments, the in vitro transcribed mRNA is a non-naturally occurring mRNA as described above.
  • the in vitro transcribed mRNA comprises a coding sequence encoding SEQ ID NO: 1 which is at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, 92% 95%, or 100% identical to SEQ ID NO: 2 (wild-type human CFTR mRNA coding sequence).
  • mRNA comprising a coding sequence encoding SEQ ID NO: 1 which is at least
  • SEQ ID NO: 2 may have greater cryptic promoter, direct and inverted repeat, and/or GC content than the mRNA discussed above. It was observed that vectors comprising SEQ ID NO: 2 frequently underwent insertion/deletion/rearrangement mutations in host cells under typical growth conditions, resulting in a heterogeneous population of vectors that could not be used directly for in vitro is found that growing host cells under conditions such as lower temperature, subdued light and/or low copy cells such as CopyCutter® reduced, but did not eliminate, the occurrence of mutation.
  • RNA comprising a coding sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%) identical to SEQ ID NO: 2 to use a template obtained by growing the vector as described above, harvesting and linearizing the vector, and purifying the desired species for use in the transcription reaction.
  • the purification step can be, e.g., size exclusion chromatography or weak anion exchange.
  • the in vitro transcribed mRNA for uses and methods according to the invention can comprise a 5'-UTR, 3'UTR, poly- A, poly-U and/or poly-C tail, cap, and/or nonstandard nucleotide residues, as discussed in the section above concerning such features.
  • compositions for use according to the invention may comprise mRNA for uses and methods according to the invention as discussed in the preceding section and additional ingredients as discussed in the section above regarding compositions comprising CFTR mRNA.
  • use and/or administration of pharmaceutical compositions comprising any of the carriers discussed above is contemplated.
  • compositions comprise PEI, such as branched PEI having a molecular weight ranging from 10-40 kDa, for example, 25 kDa.
  • compositions comprise a cationic lipid, a pegylated lipid, and an additional lipid (such as a neutral lipid).
  • the cationic lipid, pegylated lipid, and/or additional lipid may be chosen from those listed in the section above regarding compositions comprising CFTR mRNA.
  • a pharmaceutical composition as described above is administered by a route chosen from intratracheal instillation, nebulization, and aerosolization.
  • the apparatus for administering the composition can be chosen from the apparatuses listed in the section above regarding apparatuses loaded with a pharmaceutical composition.
  • the composition is administered via nebulization or aerosolization.
  • Some lipid formulations may have a tendency to aggregate when nebulization is attempted but it is generally possible to solve aggregation issues by adjusting the formulation, e.g., by substituting the cationic lipid.
  • the present invention can be used for treating cystic fibrosis.
  • the present invention provides a method of treating cystic fibrosis by administering to a subject in need of treatment an mRNA encoding a CFTR protein as described herein or a pharmaceutical composition containing the mRNA.
  • the mRNA or a pharmaceutical composition containing the mRNA may be administered directly to the lung of the subject.
  • Various administration routes for pulmonary delivery may be used.
  • an mRNA or a composition containing an mRNA described herein is administered by inhalation, nebulization or aerosolization.
  • administration of the mRNA results in expression of CFTR in the lung of the subject (e.g., epithelial cells of the lung).
  • the present invention provides a method of treating cystic fibrosis by administering to the lung of a subject in need of treatment an mRNA comprising a coding sequence which encodes SEQ ID NO: 1.
  • the present invention provides a method of treating cystic fibrosis by administering to the lung of a subject in need of treatment an mRNA comprising a coding sequence which encodes an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.
  • the present invention provides a method of treating cystic fibrosis by administering to the lung of a subject in need of treatment an mRNA comprising a coding sequence of SEQ ID NO: 3.
  • the present invention provides a method of treating cystic fibrosis by administering to the lung of a subject in need of treatment an mRNA comprising a coding sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3.
  • Additional exemplary non-naturally occurring CFTR mRNAs that can be used for treating cystic fibrosis are described in the Brief Description of Sequences section, such as, for example, SEQ ID NOs:9, 10, 11, 12, 13, 14, 15, 16, or 17.
  • non-naturally occurring CFTR mRNAs that can be used for treating cystic fibrosis comprises a coding sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 3, 9, 10, 11, 12, 13, 14, 15, 16, or 17.
  • CFTR mRNA and SNIM RNA used in the Examples disclosed herein comprised a 5' UTR with the sequence of SEQ ID NO: 4, a coding sequence (CDS) with the sequence of SEQ ID NO: 3, and a 3' UTR with the sequence of SEQ ID NO: 5.
  • FFL mRNA and SNIM RNA used in the Examples disclosed herein comprised a 5' UTR, CDS, and 3' UTR with the sequences of SEQ ID NOS: 6, 7, and 8, respectively.
  • RNA Synthesis Human cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and firefly luciferase (FFL) mRNA were synthesized by in vitro transcription from a plasmid DNA template encoding the gene, which was followed by the addition of a 5' cap structure (Cap 1) (Fechter, P.; Brownlee, G.G. "Recognition of mRNA cap structures by viral and cellular proteins" J. Gen. Virology 2005, 86, 1239-1249) and a 3' poly(A) tail of approximately 200 nucleotides in length as determined by gel electrophoresis. 5' and 3' untranslated regions were present in each mRNA product.
  • Exemplary non-naturally occurring CFTR niRNAs include SEQ ID NO:9, SEQ
  • Electrophysiological Recording Whole-cell patch-clamp recordings were conducted at room temperature using an Axopatch 200B amplifier with 5-8 ⁇ electrodes. Data were digitized (50 kHz) and filtered (5 kHz) appropriately. Series resistance was compensated (70-80%) to minimize voltage errors. Voltage-clamp recordings were performed with pipette solution of the following composition: 140 mM NMDG-Cl; 5 mM EGTA; 1 mM MgC12; 10 mM HEPES; pH 7.2; 310 mOsm/1.
  • the bathing solution contained: 140 mM NaCl, 3 mM KCl, 2 mM MgC12, 2 mM CaC12, and 10 mM HEPES; pH 7.3, adjusted to 315 mOsm/1 with D-glucose. Voltage clamp recordings commenced 3-5 minutes after establishing whole-cell configuration.
  • Cells were voltage-clamped at a holding potential of either -60 mV or 0 mV and a series of positive and negative voltage steps (either -80 mV to +80 mV or -100 to +100 mV in 20 mV increments) injected into the recorded HEK293T cells to evoke CFTR-induced whole-cell chloride (C1-) currents.
  • the membrane permeable analogue of cAMP, 8-Br-cAMP 500 ⁇ , Sigma Aldrich was applied for 4 mins to recorded cells to facilitate CFTR currents.
  • Test Compounds Test compounds were applied using a DAD-16VC fast perfusion system (ALA Scientific Instruments, USA) with the ejection pipette placed approximately 200 ⁇ from the recorded cell. 8-Br-cAMP was made as a 500 mM stock concentration in ddH20. CFTRinh-172 was made as a 10 mM stock in DMSO. All compounds were stored at -20°C and were rapidly defrosted and diluted to the desired final concentration immeHiatelv nrinr to use. [0160] Analysis. All analysis was conducted using Clampfit (MDS Analytical
  • HEK293T cells were transfected with 4 ug of hCFTR mRNA and analyzed 24 hours post transfection.
  • Whole cell clamp assays were conducted to measure current flow, as represented by chloride ion transport upon application of a set voltage.
  • a plot of current vs voltage as a result of a voltage ramp of -80 mV to +80 mV (depicted in Figure 2) demonstrates substantial differences in current when comparing untreated versus hCFTR mRNA-treated cells. This increase in current after exposure to 8-Br-cAMP, a known activator of CFTR protein, is suggestive that human CFTR protein is present in these cells.
  • CFTRinh-172 Upon treatment of these previously transfected cells with a known specific CFTR inhibitor, CFTRinh-172, the respective current drops back down to near control levels (-89% decrease). Such a decrease after exposure of this inhibitor strongly supports the presence of human CFTR protein. These results in sum demonst ate that synthetic hCFTR mRNA can produce active human CFTR protein.
  • CFTR whole cell activity assays were performed using an automated system (Ion Works) within HEK293 cells. As described above, treated cells as well as control cells (untreated and mock trans fected) were subjected to activator and inhibitor substrates to help determine changes in current flow (chloride ion transport).
  • GlyH-101 is believed to act as a CFTR pore blocker which acts upon the extracellular membrane side of the protein. Notably, this action of mechanism is different from that of CFTRinh-172, which is reported to function from the intracellular side of the CFTR protein.
  • DOPE, Choi and DMG-PEG2K were mixed and diluted with ethanol to 3 mL final volume.
  • an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of CFTR mRNA was prepared from a 1 mg/mL stock.
  • the lipid solution was injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol.
  • the resulting nanoparticle suspension was filtered, diafiltrated with lx PBS (pH 7.4), followed by water, concentrated and stored at 2-8°C.
  • Final concentration 1.09 mg/mL CFTR mRNA
  • FFL and CFTR protein produced via intratracheal administered mRNA-loaded nanoparticles All studies were performed using either female BALB/C mice or CFTR KO mice.
  • FFL samples were introduced via either direct instillation (Micro Sprayer®) or nebulization (PARI Boy or Aeroneb) respective dose of encapsulated FFL mRNA.
  • CFTR mRNA was introduced using a PARI Boy jet nebulizer. Mice were sacrificed and perfused with saline after allowing time for expression.
  • FFL test materials were administered by a single intratracheal aerosol administration via a MicrosprayerTM (50 while animals are anesthetized with intraperitoneal injection of a mixture of ketamine 50-100 mg/kg and xylazine 5-15 mg/kg.
  • Nebulization Administration of FFL mRNA.
  • PEI Polyethyleneimine
  • CFTR KO mice were treated with polyethyleneimine (PEI)-based CFTR mRNA loaded nanoparticles via intratracheal administration (30 ug encapsulated mR A). The treated mice were sacrificed six hours and twenty-four hours post-administration and the lungs were harvested and fixed in 10% neutral buffered formalin (NBF). In situ hybridization was employed for detection of the exogenous human CFTR mRNA ( Figure 6). Substantial staining was observed 24 hours post- administration with widespread distribution in both mouse lungs of the treated CFTR KO mice while no staining was observed for PBS-treated control mice.
  • PEI polyethyleneimine
  • CFTR mRNA-loaded cationic lipid nanoparticles utilizing CI 2-200 as the cationic lipid component.
  • Protein lysates were prepared from human bronchial epithelial cell line (BEAS-2B), human embryonic kidney cell line (HEK), mouse lungs and pig lungs using ProteoExtract Transmembrane Kit (Merck) and transmembrane fraction used for immunoblotting using different primary antibodies (MAI -935 from Thermo Scientific Pierce Antibodies, Rockford, IL, USA, AB596 from the Cystic Fibrosis Consortium, University of Pennsylvania, PA, USA, and AB570 from the Cystic Fibrosis Consortium, University of Pennsylvania, PA, USA). The data are summarized as Figure 13.
  • HGT5001 Formulation Aerosol experiments using hCFTR SNIM RNA in a formulation of HGT5001 :DOPE;Chol;PEGDMG2K (relative amounts 50:25:20:5
  • IP results using tissue material from pigs provided the evidence for the technical feasibility of hCFTR detection post transcript delivery in vivo.
  • the antibodies used in immunoprecipitating CFTR (R29, R66/17 and R66/16) are commercially available, other commercially available antibodies were screened for their efficacy in IP reactions.
  • Aerosol administration of firefly luciferase (FFL) SNIM RNA to the pig lungs was established by a stepwise experimental procedure.
  • FFL SNIM RNA formulations were nebulized to anaesthetized pigs during controlled ventilation.
  • lungs were excised immediately after aerosol administration was completed and lung specimens were incubated in cell culture medium overnight before ex vivo luciferase measurement was performed on lung specimens by BLI.
  • the pigs had a body weight ranging from 35-90 kg. Each treatment was performed on one pig. In total five pigs were treated.
  • the first pig (90 kg weight) was treated with FFL SNIM RNA in the PEI Formulation of Example 6 using an EFlow mesh nebulizer and measurement of luciferase activity in lung homogenates.
  • the second pig (60 kg weight) was treated with FFL SNIM RNA in the PEI Formulation of Example 6 using an EFlow mesh nebulizer and measurement of luciferase activity in lung specimens by BLI.
  • the third pig (80 kg weight) was treated with FFL SNIM RNA in the PEI Formulation of Example 6 using a PARI BOY jet nebulizer and measurement of luciferase activity in lung specimens by BLI.
  • the fourth pig (60 kg weight) was treated with FFL SNIM RNA/hCFTR mRNA in the PEI
  • Example 6 Formulation of Example 6 using an Aeroneb mesh nebulizer and measurement of luciferase activity in lung specimens by BLI.
  • the fifth pig 35 kg weight was treated with FFL SNIM RNA in the HGT5001 Formulation of Example 6 using an Aeroneb mesh nebulizer and measurement of luciferase activity in lung specimens by BLI.
  • tissue specimens were collected from various lung regions followed by incubation in cell culture medium overnight.
  • tissue specimens were either homogenized and analyzed in a tube luminometer or incubated in a medium bath comprising D-Luciferin substrate and subjected to ex vivo luciferase BLI.
  • EFlow mesh nebulizer was connected in-line to the ventilation tubing of the respirator. Aerosol administration took approximately 60 min and was longer than expected from control experiments with an open system. This was apparently caused by increased back pressure during nebulisation as evidenced by aerosol outflow at the reservoir of the mesh nebulizer.
  • Eight milliliters of the PEI Formulation of Example 6 comprising 1 mg FFL SNIM RNA in water for injection were prepared as described in WP5 and were nebulized in two separate 4 ml portions one after another. Luciferase measurement was performed in tissue homogenates of excised lung specimens of various lung regions after overnight incubation in cell culture medium. Expression values were mapped according to the origin of the lung specimens ( Figure 21).
  • Luciferase expression was highest in central parts of the lung and declined towards more distal regions of the lung.
  • the expression pattern correlated with the expected deposition pattern of the inhaled FFL SNIM RNA-PEI nanoparticles according to the chosen ventilation parameters.
  • Levels of luciferase expression were in the same range as observed in mouse experiments in WP5 using the same the PEI Formulation of Example 6.
  • PEI Formulation of Example 6 in pig #2 was performed as in pig #1 but luciferase activity was measured on lung specimens by bioluminescent imaging (BLI). This experiment was performed to establish ex vivo luciferase measurement of organ cultured lung specimens by BLI.
  • pig #3 was treated using the PARI BOY jet nebulizer which was connected to the ventilation tubing via a T-connector. Aerosol administration lasted longer (approximately 80 min) than with the EFlow mesh nebulizer and aerosol administration was non-satisfying. Very low luciferase activity was detected in sliced lung samples from different lung regions of the treated pig ( Figure 23).
  • the experiment confirmed results obtained from pig #1 and pig #2, although higher expression levels were obtained.
  • the experiment showed that the Aeroneb mesh nebulizer was best suited for delivery of the the PEI Formulation of Example 6 to the lungs of pigs.
  • the experiment demonstrated FFL SNIM RNA was still active when co- delivered together with hCFTR mRNA.
  • Pig #5 was treated with 1 mg of FFL SNIM RNA in the HGT5001 Formulation of Example 6 aerosolized with the Aeroneb mesh nebulizer.
  • the formulation could be aerosolized without technical difficulties. Luciferase activity was clearly observed in individual tissue specimens of different lung regions of the treated pig ( Figure 25).
  • the experiment showed that aerosolized FFL SNIM RNA in the HGT5001
  • Example 6 is active in pig lung tissue, although expression levels were approximately 15-20-fold lower than in pigs treated with the the PEI Formulation of Example 6.
  • Anesthesia was maintained with continuous intravenous infusion of 1% propofol as required. Ventilation parameters were matched with endexpiratory carbon dioxide and adjusted if necessary. Anesthesia, respiratory and cardiovascular parameters were monitored continuously using pulse oximetry, capnography, rectal temperature probe and reflex status. Animals received infusion of balanced electrolyte solution at 10 ml/kg/h. Duration of the anesthesia was approximately 80-120 min. Pigs were extubated after onset of sufficient spontaneous breathing. Pigs were killed with bolus injection of pentobarbital 100 mg/kg of body weight via the lateral ear vein after sedation. Lungs were excised and sliced approximately 1 cm thick tissue specimens were collected from various lung regions followed by incubation in cell culture. For measurement of luciferase activity tissue specimens were incubated in a medium bath comprising D-Luciferin substrate and subjected to ex vivo luciferase BLI.
  • Example 6 Formulation of Example 6 on day 1 and 1 mg FFL SNIM RNA and 1 mg hCFTR SNIM RNA in the PEI Formulation of Example 6 on day 8), Luciferase activity was clearly detected in lung specimens of twice-treated pigs #4 and #8 ( Figure 28). Luciferase expression was highest in central parts of the lungs. It has to be considered that samples were stored for additional 10 hours in cell culture medium before measurement because of a power blackout on the day of the
  • Example 6 Formulation of Example 6 on day 1 and day 8, aerosol administration of 1 mg FFL SNIM RNA and 1 mg hCFTR SNIM RNA in the PEI Formulation of Example 6 on day 15), Luciferase activity was clearly detected in lung specimens of thrice-treated pigs #1 and #2 ( Figure 29). Luciferase expression was highest in central parts of the lungs.
  • Toxicology Adverse events. kind and frequency of adverse events (AE) were analyzed by laboratory parameters (blood, MBS and BAL) and by physical examination of the pigs (defined as a secondary objective in this trial).
  • Serum and whole blood samples were taken at the time points defined by the study protocol. Twelve representative parameters (haemoglobin, hematocrit, AP, ALT, AST, CK, bilirubin, creatinine, glucose, potassium, thrombocytes, and white blood cells) being indicative to show organ specific pathology (blood, bone marrow, liver, muscle, and kidney) were selected and the test results obtained from the VetMedLab, Ludwigsburg, Germany classified according to VCOG, version 2011.
  • BALF Brocho-Alveolar-Lavage fluid
  • MFS microbiology samples
  • a CFTR protein may be effectivey expressed from a CFTR encoding mRNA with a signal peptide encoding sequence.
  • Human embryonic kidney HEK293T cells were grown in DMEM (Invitrogen Cat # 11965-092) supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin. The day before transfection, cells were plated on 6-well plates at 50-60% confluence and incubated under normal tissue culture conditions (36°C in a humidified atmosphere of 5% C02, 95% air). In preparation for transfection, 60 ⁇ Lipofectamine 2000 (Invitrogen Cat # 11668019) was diluted in OptiMem reduced serum media (Invitrogen Cat # 31985-062) and gently vortexed.
  • DMEM Invitrogen Cat # 11965-092
  • OptiMem reduced serum media Invitrogen Cat # 31985-062
  • Lipofectamine ® and incubated at room temperature for 30 minutes. The plating media was gently aspirated and replaced with 1 ml OptiMem Reduced Serum Medium and 300 ⁇ of each respective mRNA/Lipofectamine ® complex. Cells were incubated under standard tissue culture conditions.
  • the required amount of SNIM RNA was diluted just before application in water for injection (Braun, Melsungen) to a total volume of 4 ml and added quickly to 4 ml of an aqueous solution of branched PEI 25 kDa using a pipette at an N/P ratio of 10.
  • the solution was mixed by pipetting up and down ten times and nebulized as two separate 4.0 ml fractions one after another to the mouse lungs using the indicated nebulizer.
  • cKK-E12 Formulation For the lipid-based nanoparticle experiment, a lipid formulation was created using CO-CFTR-C-HislO SNIM RNA in a formulation of cK - E12:DOPE:Chol:PEGDMG2K (relative amounts 50:25:20:5 (mg:mg:mg:mg). The solution was nebulized to the mouse lungs using the indicated nebulizer.
  • Nebulization Administration of Human CO-CFTR-C-HislO mRNA.
  • CFTR test materials were administered by a single aerosol inhalation via PARI Boy jet nebulizer (nominal dose volume of up to 8 mL/group).
  • CFTR mRNA was prepared in the manner described above. Four CFTR knockout mice were placed in an aerosol chamber box and exposed to 2 mg total codon optimized unmodified human CFTR mRNA (comprising the coding sequence of SEQ ID NO: 3) via nebulization (Pari Boy jet nebulizer) over the course of approximately one hour. Mice were sacrificed 24 hours post-exposure.
  • Aerosol administration of a combination of firefly luciferase (FFL) SNIM RNA and codon optimized human CFTR (CO-CFTR) SNIM RNA at varying concentrations to pig lungs was established by a stepwise experimental procedure.
  • FFL/CO-CFTR SNIM RNA formulation was nebulized to anaesthetized pigs during controlled ventilation.
  • the animals were sacrificed by bolus injection of pentobarbital (100 mg/kg of body weight) and potassium chloride via the lateral ear vein after sedation 24 hours after aerosol administration was completed. Lungs were excised and sliced to approximately 1 cm thick tissue specimens.
  • tissue specimens were incubated in a medium bath comprising D-Luciferin substrate and subjected to ex vivo luciferase BLI. After BLI, samples from luciferase-positive and luciferase-negative regions were taken for histopathology, immunohistochemistry and in situ hybridization. The residual specimens were shock-frozen in liquid nitrogen and subsequently stored at -80°C until analysis by IP/WB and Elisa.
  • the pigs had a body weight ranging from 35-90 kg.
  • the study was designed using both age and weight-matched pigs to control for variability.
  • a single cohort of 6 pigs (3 male and 3 female) was established for each experimental group of the 4-arm study.
  • the first cohort was treated with water for injection (WFI) alone, which was administered using a Aeroneb mesh nebulizer.
  • the second cohort was treated with a solution of 1 mg FFL SNIM RNA and 1 mg of codon optimized human CFTR (CO-CFTR) SNIM RNA in the PEI Formulation described below, using an Aeroneb mesh nebulizer.
  • WFI water for injection
  • CO-CFTR codon optimized human CFTR
  • the third cohort received 1 mg of FFL SNIM RNA and 5 mg of codon optimized human CFTR (CO-CFTR) SNIM RNA in the PEI Formulation described below.
  • the fourth cohort was treated with 1 mg of FFL SNIM RNA and 10 mg of codon optimized human CFTR (CO-CFTR) SNIM RNA in the PEI Formulation described below.
  • the scheme for treatment and evaluation of each group is shown in Table 4 below. Table 4.
  • mRNA - PEI formulation An exemplary standardized formulation procedure described below was performed just before treatment of the animals.
  • SNIM RNA and 1 mg FFL SNIM RNA N/P 10 in a volume of 8mL: 3mL water for injection and 3mL RNA stock solution (c: lmg/mL in water; 1.5mL FFL mRNA + 1.5mL CFTR mRNA) were filled into a 15mL falcon tube.
  • RNA stock solution c: lmg/mL in water; 1.5mL FFL mRNA + 1.5mL CFTR mRNA
  • a 15mL falcon tube In a second falcon tube 5.61mL water for injection were mixed with 0.39mL brPEI stock solution (c: lOmg/mL in water).
  • Two 20mL syringes were fixed in the mixing device. Each of them was connected to a needle via a tubing.
  • One syringe was filled with the RNA- and the other with the PEI-solution using the withdrawal function of the syringe pump. (Settings: Diameter: 20.1mm, Flow: 5mL/min, Volume: 5.9mL). The needles were removed and the tubes connected to the mixing valve. It was important to connect the syringe containing the RNA-solution to the angled position of the valve. To control the outlet diameter, a needle was connected. The mixing was performed using the infusion function of the syringe pump (Settings: Diameter: 20.1mm, Flow: 40mL/min, Volume: 5.8mL). To achieve a reproducible polydispersity index, the samples were fractionated manually during mixing.
  • Table 5 Exemplary volumes and settings for different mixing volumes
  • V(withdrawal) and V(infusion) designate the setting on the syringe pump for aspiration and dispension, respectively, of the mRNA and PEI components.
  • protein lysates were prepared using the same protocol and buffers used for animal tissues with exclusion of homogenization step. Cells from three wells were pooled for analysis. Expression of human CFTR was detected using immunoprecipitation with R24.1 antibody (R&D Systems) and Western Blot with a combination of 217, 432 and 596 antibodies (all from Cystic Fibrosis Consortium, University of Pennsylvania, PA, USA). hCFTR could be detected for all of complexes nebulized in pigs (see Figures 54-57).
  • Aerosol Application The aerosol (WFI alone 44 ml; modified mR A PEI formulation in WFI: 8, 24 and 44 ml) was nebulized and inhaled into the anaesthetized pig via an Aeroneb ® mesh nebulizer. Sedation in pigs was initiated by premedication with azaperone 2 mg/kg body weight, ketamine 15 mg/kg body weight, atropine 0.1 mg/kg body weight and followed by insertion of an intravenous line to the lateral auricular vein. Pigs were anesthetized by intravenous injection of propofol 3-5 mg/kg body weight as required.
  • Anesthesia was maintained by isoflurane (2-3%) with 1% propofol bolus injection at 4 to 8 mg/kg body weight to enhance anesthesia as required. Duration of the anesthesia was approximately 1-3 hrs. Pigs were sacrificed with bolus injection of pentobarbital (100 mg/kg body weight) and potassium chloride via the lateral ear vein 24 hours after completion of aerosolization. Lungs were excised and tissue specimens were collected from various lung regions. The stored samples were subjected to different assessment methods such as bioluminescence, histopathology, IP/Western Blot and Elisa.
  • CFTR Expression Analysis by Western Blot and Immunohistochemistry FFL positive tissues samples were excised (minimum of 10 samples for each pig within a cohort) and analyzed by immuneprecipitation / Western blot (IP-WB) and immunohistochemistry for human CFTR. Briefly, protein lysates were prepared from pig lungs as follows: Between 300-400mg of lim p tissue was used for analysis. The tissue was homogenized in basis buffer (20mM Tris, 150 mM NaCl, pH 8.0) containing protease inhibitors using LysingMatrixA (MPBiomedicals, Ref:6910-500) and Homogeniser "FastPrep24" (MP Biomedicals).
  • CFTR-positive bronchi and bronchioles A bronchus / bronchiole was regarded as positive if at least one epithelial cell was detected within the epithelial cell layer displaying a clear membrane- localizaed CFTR signal. A representative image of a "positive" sample is depicted in Figure 40. Coditions for CFTR immunohistochemistry were optimized by assessing specificity of available antibodies against CFTR utilizing single antibody or combinations of up to three antibodies respectively. Clear CFTR- specific signals were observed after incubation of antibody 596.
  • RNAscope® Advanced Cell Diagnostic
  • CFTR specific RNA probe The hCFTR probe was shown bind CFTR, with cross reactivity to human, mouse, rat, pig and monkey. Once bound, the probe is hybridized to a cascade of signal amplification molecules, through a series of 6 consecutive rounds of amplification.
  • the sample dth an HRP-labeled probe specific to the signal amplification cassette and assayed by chromatic visualization using 3,3'-diaminobenzidine (DAB).
  • a probe specific for Ubiquitin C was used as the positive control ( Figures 47A and 48A), while dapB was used as the negative control ( Figures 47B and 48B). Positive CFTR signal was compared to that of untreated and vehicle control treated porcine lung tissue ( Figure 49 A and B).
  • F/samnle M In Vivo Expression in the Lung
  • This example desmonstrates successful in vivo expression in the lung following aerosol delivery of mRNA-loaded nanoparticles. All studies were performed using pigs of the German Landrace, obtained from Technical University Kunststoff, Weihenstephan, Germany. The pigs had a body weight ranging from 35-90 kg. FFL/CO-CFTR-C-HislO mRNA formulation or vehicle control was introduced using a Pari jet nebulizer. Pigs were sacrificed and perfused with saline, after a predetermined period of time, to allow for protein expression from the mRNA.
  • cKK-E12 Formulation For the lipid-based nanoparticle experiment, a lipid formulation was created using 1 mg FFL + 9 mg of CO-CFTR-C-HislO mRNA encapsulated in a formulation of cKK-E12:DOPE:Chol:PEGDMG2K (relative amounts 40:30:25:5 (mol ratio). The solution was nebulized to the Pig lungs using the indicated nebulizer.
  • Aerosol Application The aerosol (Saline or CO-FFL cK -E12 formulation) was nebulized and inhaled into the anaesthetized pig. Sedation in pigs was initiated by premedication with azaperone 2 mg/kg body weight, ketamine 15 mg/kg body weight, atropine 0.1 mg/kg body weight and followed by insertion of an intravenous line to the lateral auricular vein. Pigs were anesthetized by intravenous injection of propofol 3-5 mg/kg body weight as required.
  • Anesthesia was maintained by isoflurane (2-3%) with 1% propofol bolus injection at 4 to 8 mg/kg body weight to enhance anesthesia as required. Duration of the anesthesia was approximately 1-3 hrs. Pigs were killed with bolus injection of pentobarbital (100 mg/kg body weight) and potassium chloride via the lateral ear vein. Lungs were excised and tissue specimens were collected from various lung regions followed by incubation in cell culture medium overnight. The stored samples were subjected to bio luminescence detection.
  • tissue specimens were either homogenized and analyzed in a tube luminometer or incubated in a medium bath comprising D-Luciferin substrate and subjected to ex vivo luciferase BLL A strong
  • SEQ ID NO 1 Wild-type CFTR amino acid sequence.
  • SEQ ID NO 2 Wild-type CFTR mRNA coding sequence.
  • SEQ ID NO 14 Non-naturally occurring CFTR mRNA coding sequence #7.

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